The goal of this project is to determine the DNA sequence of the chromosome of Treponemia pallidum, the causative agent of syphilis. This important human pathogen cannot be cultured outside of animals and has been a challenging organism to study. During the last decade the number of cases of syphilis has doubled after nearly 40 years of declining incidence. It has been particularly difficult to apply molecular genetic methods to T. pallidum because of the inability to cultaure the organism in vitro. It is clear that a genomic DNA sequence and a corresponding set of mapped recombinant clones would provide a tremendous resource for the study of this organism. Previously we showed that the genome of T. pallidum is relatively small, about 1Mb in size. We constructed a restriction enzyme cleavage map of the genome, showing that it is a circular chromosome, and we mapped over 100 landmarks throughout the chromosome. Determining the DNA sequence of the T. pallidum genome is the next logical step in this analysis. With the recent developments in DNA sequence of the T. pallidum genome is the next logical step in this analysis. With the recent developments in DNA sequencing technology, and the application of these advances to determination of other bacterial genomes, a T. pallidum genome project is now feasible. The approach will be a moderate redundancy, complete coverage DNA sequence of the genome. This will be accomplished by sequencing the ends of inserts in a large set (e.g. 20,000) of plasmid clones. This will not only produce the genomic sequence, but also will provide a mapped set of small insert clones spanning the genome as well as a set of fusions of the lac promoter (present in the vector) to each gene of the chromosome. The former clone set will be useful for studies of other Treponema strains while the latter will provide a means for heterologous expression of all T. pallidum genes. It will also be possible to identify similarities between T. program). This will provide a "parts list" for the organism. This approach gives immediate answers to a number of important questions. For example, it will be possible to identify which metabolic activities are present in T. pallidum, since sequence similarities are well conserved among these proteins. This in turn may lead to an understanding of which metabolic functions are lacking in T. pallidum, and the development of a medium for in vitro growth. In addition, functions that have sequence similarity to known virulence factors will be identified. These can be readily targeted for further study since the relevant clones will be available. A genome database will also be developed for this project, which will be available on the Internet. This will allow all researchers to access the large of amount of information on sequences, functions, clones, and other physical map features that will be generated.